The present invention relates to a recombinant bacteriophage, pseudovirion or phagemid that is capable of entering bacteria by specific binding to an artificial receptor. Said receptor does not comprise at its active binding site elements such as proteins or peptides that are derived from the natural receptor used in the specific initial bacteriophage-bacterium interaction.
Bacteriophages, like bacteria, are very common in all natural environments. Bacteriophages (phages) are intracellular parasites. Bacteria and their phages have a common evolutionary history and phages may have adapted to their host species by multiple mechanisms. The phage genome may consist of double-stranded DNA, single-stranded DNA, double-stranded RNA or single-stranded RNA. Bacteriophages exist in several morphologies and can be spherical, cubic, filamentous, pleomorphic or tailed. Based on their life cycle, bacteriophages can be divided into three groups: the virulent phages capable of only lytic propagation (called lytic phages), the so-called temperate phages capable of either lytic propagation or lysogenic phase and the non-lysing phages where the mature phage is continuously extruded. The virulent life cycle of wild type phages consists of infection of the host cell, i.e. attachment to a specific receptor in the bacterial cell wall, followed by entering of the phage genome in the cell, replication of the phage genome, production of the phage structural components, phage assembly and release of the progeny phages after lysis of the host cell. In the lysogenic life cycle, the phage genome exists as a prophage resulting in coexistence of phage and host cell without lysis. Usually, this is achieved by integration of the phage genome into the bacterial chromosome. The life cycle of the non-lysing phages, like e.g. Bacteriophage M13, is similar to that of the lytic phages, but the infection is not followed by lysis. Bacteriophages have been extensively used in biotechnology. Phage genes or complete phages may be used to obtain lysis and/or killing of bacteria.
U.S. Pat. No. 4,637,980 describes the use of an E. coli strain containing defective temperature sensitive lambda lysogens as a method for cell disruption. Smith and coworkers (Smith et al., 1987, J. Gen Microbiol. 133; 1111-1126) describe the use of bacteriophages to treat diarrhea in calves, caused by seven different bovine enteropathogenic strains of E. coli WO95/27043 describes a method to treat infectious diseases caused by several bacterial genera, such as Mycobacterium Staphylococcus, Vibrio, Enterobacter, Enterococcus, Eschericia, Haemophilus, Neisseria, Pseudomonas, Shigella, Serratia, Salmonella and Stretococcus, comprising the administration of bacteriophages with delayed inactivation by the animal host defence system. WO98/51318 describes a diagnostic kit and a pharmaceutical composition, comprising bacteriophages to diagnose and to treat bacterial diseases caused by bacteria, such as Listeria, Klebsielia, Pneumococcus, Moraella, Legionelle, Edwardsiella, Yersinia, Proteus, Heliobacter, Salmonella, Chlamrydia, Aeromonas and Renibacterium.
Another application of bacteriophages is the in vitro selection of proteins displayed on the tip of filamentous phages on immobilised target (=biopanning), which is a powerful technique for the isolation of interacting protein-ligand pairs from large libraries, such as antibody libraries (for a recent review: Rodi and Makowski, 1999, Curr. Opin. Biotechn., 10: 87-93). However, for optimal in vitro biopanning, a purified target protein is needed. Moreover, high quality of the library is a prerequisite for success. Enrichment for selfligated vector, phages carrying incomplete sequences, incorrect reading frames, deletions and amber stop codons are very often observed (Beekwilder et al, 1999, Gene, 228, 23-31 and de Bruin et al, 1999, Nature Biotechnology, 17: 397-399). In the search to avoid the problems encountered with panning using imperfect libraries, several alternative techniques, both bacteriophage based and non bacteriophage based, have been developed. Non bacteriophage based techniques are, amongst others ribosome display (Dall""Acqua and Carter, 1998, Curr. Opin. Struct. Biol., 8: 443-450) and the yeast two-hybrid system (Drees, 1999, Curr. Opin. Chem. Biol., 3: 64-70). Bacteriophage based techniques comprise display on phage lambda, SIP (Spada and Pluckthun, 1997, Biol. Chem., 378: 445-456; EP0614989) and CLAP (Malmborg et al, 1997, J. Mol. Biol., 273: 544-551; WO9710330). SIP and CLAP are in vivo selection techniques and have the advantage that the F+E.coli host cells can only be infected by bacteriophages or pseudovirions when a matched pair is formed. Both systems are based on the fact that pilin on the F-pili of E.coli cells serve as the natural receptor for binding of the D2-domain of pill from the phage (Deng et al., 1999, Virology, 253:271-277). This results in retraction of the pilus, so that an interaction between the D1 domain of pill with the TOL protein complex located in the E.coli cell membrane leads to the infection (Deng et al, 1999, Virology, 253: 271-277). SIP has the disadvantage that it only works for high affinities of the binding pairs and that each target needs to be cloned, expressed and purified as a fusion with the D2 domain of pill. Therefore, with SIP, normally only one target can be screened at the time. For CLAP only small peptides (15-18 amino acids) can be expressed on the F-pilus, hence, this technique can only be used for small linear epitopes. An additional disadvantage is the need for modified M13 to avoid natural infection of host cells. Therefore, removal of the D2 domain of pill is essential. This results in a truncated form of M13 and concomitant difficulties to obtain good titres.
It is known that bacteriophages use specific receptors on the host cell wall as a way to recognise the host cell and to start the infection process. In all the applications cited above, the propagation of phages, pseudovirions or phagemids is dependent on the use of the natural phage receptor, or part of it, on the host cell wall. For M13, mainly used in these systems, the natural receptor is pilin (Malmborg et al., 1997, J. Mol. Biol. 273: 544-551). Other examples of natural receptors are lamB for bacteriophage lambda (Werts et al, 1994, J. Bacteriol. 176: 941-947), the outer membrane protein OmpA for bacteriophages K3, Ox2 and M1 (Montag et al, 1987, J. Mol. Biol., 196: 165-174), the outer membrane proteins OmpF and Ttr for bacteriophage T2 (Montag et al, 1987, J. Mol. Biol., 196, 165-174), the outer membrane protein OmpC for the T4 phage family (T4, Tula, Tulb) (Montag et al.,1990, J. Mol. Biol., 216: 327-334). The T4 bacteriophage family is using a C-terminal region of protein 37 as natural ligand (Montag et al., 1990, J. Mol. Biol., 216: 327-334), bacteriophages T2, K3, Ox2 and M1 are using protein 38 as natural ligand (Montag et al, 1987, J. Mol. Biol., 196, 165-174) whereas phage lambda is using the C-terminal portion of the lambda tail fibre protein as natural ligand (Wang et al., 1998, Res. Microbiol, 149: 611-624). Bacteriophagexe2x80x94receptor independent phage binding to mammalian cells expressing the growth factor receptor ErbB2 followed by receptor mediated endocytosis was also described: Marks and collaborators (Poul and Marks, 1999, J. Mol Biol., 288: 203-211 and Becceril and Marks, 1999, Biochem. Biophys. Res. Commun., 255: 386-393) successfully isolated phage capable of binding mammalian cells expressing the growth factor receptor ErbB2 and undergoing receptor mediated endocytosis by selection of a phage antibody library on breast tumour cells and recovery of infectious phage from within the cell. However, the phage could not propagate in the mammalian cell, and the detection of the cells carrying bacteriophage could only be realised in an indirect way, by expression green fluorescent protein as a reporter gene.
One aspect of the invention is a genetically modified bacteriophage, pseudovirion or phagemid that is not dependent upon its natural receptor or parts thereof for entering a host cell.
Another aspect of the invention is a genetically modified bacteriophage, pseudovirion or phagemid capable of entering a host cell by specific binding to an artificial receptor. These artificial receptors can be endogenous host cell proteins located at the bacterial surface, or parts thereof, that are normally not involved in the bacteriophagexe2x80x94bacterium interaction, but it may also be heterologous proteins, preferentially fusion proteins displaying an oligo- or polypeptide on the bacterial surface. The genetically modified bacteriophage, pseudovirion or phagemid binds to the artificial receptor preferentially by an artificial ligand. A specific embodiment is a genetically modified bacteriophage that is not dependent upon OmpA, OmpC, OmpF, Ttr or pilin for interaction with and/or entering E.coli. A further specific embodiment is a genetically modified M13 bacteriophage, pseudovirion or phagemid that does not depend upon pilin, or fragments thereof for specific interaction with and/or entering of E. coli. Said M13 bacteriophage, pseudovirion or phagemid can enter,both F+and Fxe2x88x92E. coli cells, dependent upon an artificial receptor that is displayed on the surface of said cells.
Still another aspect of the invention is a bacteriophage, pseudovirion or phagemid that enters the host cell mediated by an antigenxe2x80x94antibody reaction, whereby in the binding complex no proteins or parts of the natural receptor are involved.
A preferred embodiment of the invention is a genetically modified M13 phage, pseudovirion or phagemid displaying an antibody, preferentially the variable part of a camel heavy chain antibody for instance disclosed in international patent application WO94/04678 and in Hamers-Casterman C et al Nature,vol 363, Jun. 3, 1993.p 446-448, on its tip, which can enter an E.coli host cell, displaying the antigen, preferentially as an pOprl fusion protein. The use of Oprl as a protein for the expression of an amino acid sequence at the surface of the cell wall of a host cell is disclosed for example in international patent application WO95/04079 which is incorporated herewith by reference.
A further aspect of the invention is the use of said bacteriophage, pseudovirion or phagemid for selective entering of a subpopulation of bacteria. Using the specific artificial receptor interaction, in a mixed culture, the bacteriophage, pseudovirion or phagemid will only enter those bacteria that carry said artificial receptor. By this, the subpopulation of bacteria can be identified and/or eliminated. One embodiment of the invention is the specific elimination of pathogenic bacteria by directing a recombinant bacteriophage, pseudovirion or phagemid to a specific bacterial surface protein of said pathogenic bacteria. The pathogenic bacteria can be gram positive, gram negative or gram variable and can belong, amongst other to the genera Aeromonas, Chiamydia Edwardsiella, Enterobacter, Enterococcus, Eschedchia, Haemophilus, Heliobacter, Klebsiella, Legionella, Listeria, Moraxella, Mycobacterium, Neisseria, Pneumococcus, Proteus, Pseudomonas, Renibacterium, Salmonella, Sernatia, Shigella, Staphylococcus, Vibrio or Yersinia, without that this summation is limitative.
Elimination can be obtained by the lytic cycle of the bacteriophage, but is not limited this method. Other methods of eliminating the host cell may be the production of a toxic product encoded by the recombinant bacteriophage genome in the host cell. A preferred embodiment is the production of barnase placed after an inducible promoter, such as the barnasexe2x80x94barstar cassette described by Jucovic and Hartley (Protein engineering, 8: 497-499, 1995).
Another aspect of the invention is a host cell, entered by the genetically modified bacteriophage, pseudovirion or phagemid. Such host cell comprises the nucleotide sequence encoding the artificial receptor and the nucleotide sequence encoding the artificial ligand. Such sequences may be expressed in the host cell in combination with marker sequences, especially sequences encoding antibiotic resistance genes. A preferred embodiment is an E. coli cell, preferentially transformed with a plasmid encoding a pOprl-fusion protein, more preferentially transformed with a plasmid derived from ptrc-Oprl, carrying a genetically modified M13 phage, pseudovirion or phagemid, preferentially a pK7C3 derived phagemid, wherein said genetically modified M13 phage is modified, especially by in vitro construction, with a nucleotide sequence encoding a protein capable of specifically binding to the pOprl-fusion protein.
In a particular embodiment of the invention, the Oprl-fusion protein is carried out in introducing the nucleotide sequence of the fusion partner acting as the region for interaction with the ligand expressed on the bacteriophage, pseudoviron or phagemid, especially as disclosed in WO95/04678.
Still a fiber aspect of the invention is the use of said bacteriophage, pseudovirion or phagemid to identify interacting proteins, including cases where none of the members of the interacting protein is kaown.
In different embodiments, the bacteriophage, pseudovirion or phagemid can be used to screen (1) a host cell, displaying a bait against a library of bacteriophages, pseudovirions or phagemids displaying the prey, (2) a bacteriophage, pseudovirion or phagemid displaying a bait against a library of host cells displaying the prey, (3) a library of bacteriophages, pseudovirions or phagemids displaying different preys or baits against a library of host cells, displaying different baits or prey (As illustrated in FIG. 1).
A preferred embodiment is where pOprl is used as fusion partner for the display of bait or prey on the surface of the Fxe2x88x92E.coli strains (Williams and Meynell 1971. Mol. Gen. Genet. 113: 222-227) such as DH5xcex1 and UT5600 as host cell and where the phagemid pK7C3 is used for cloning the prey or bait as a pill fusion protein.
Another embodiment of the invention is the construction of a subtraction library, with the use of lytic bacteriophages, preferentially barnase expressing bacteriophages. In this embodiment, a part of the host cell library is recognised by lytic phages such as barnase expressing phages and killed upon recognition of the artificial receptor by the artificial ligand, entering of the bacteriophage, pseudovirion or phagemid and expression of the lytic gene. Another aspect of the invention is a method for selecting artificial receptorxe2x80x94artificial ligand interactions, comprising
growing a host cell or a mixture of host cells displaying one or more artificial receptors,
contacting said host cell or said mixture with a genetically modified bacteriophage, pseudovirion or phagemid or a mixture of genetically modified bacteriophages, pseudovirions or phagemids with one or more artificial ligands,
selecting those cells that have been entered by one or more bacteriophages, pseudovirion of phagemid.
One embodiment of the invention is said method, whereby the selection is based on an antibiotic resistance marker. Another embodiment is said method whereby the cells are selected by killing of the host cell, preferentially by expression of barnase. A preferred embodiment is said method, whereby the host cell is an E. coli cell, displaying the artificial receptor as a pOprl fusion protein, and the genetically modified bacteriophage, pseudovirion or phagemid is a genetically modified M13, displaying an artificial ligand as a pill fusion protein.
The following definitions are set forth to illustrate and define the meaning and scope of the various terms used to describe the invention herein.
Genetically modified bacteriophage: a bacteriophage of which the genome has been modified, at least by the introduction of the gene encoding for an artificial ligand. This introduction can be as a replacement of one of the endogenous genes or as an additional gene besides the endogenous genes.
Natural receptor: protein domain, protein or protein complex situated on the host cell wall, involved in the natural initial interaction between a bacteriophage and said host cell, whereby this interaction is followed by introduction of the genetic material of the bacteriophage into the host cell.
Artificial receptor: protein domain, protein, fusion protein or protein complex on the host cell wall whereby said protein domain, protein, fusion protein or protein complex does not contain one or more peptide fragments of at least 10 contiguous amino acids derived from the natural bacteriophage receptor in the protein sequence or region that is involved in the interaction between bacteriophage, pseudovirion or phagemid and the artificial receptor.
Protein : encompasses peptide, protein, glycoprotein, lipoprotein or another form of modified protein, including chemically modified protein.
Protein complex: proteinxe2x80x94protein complex, but also proteinxe2x80x94compound complex, whereby said compound may be any chemical or biological compound, including simple or complex inorganic or organic molecules, peptido-mimetics, carbohydrates, nucleic acids or derivatives thereof.
Natural ligand: protein, protein domain or protein complex of the bacteriophage, pseudovirion, or phagemid involved in the natural initial interaction between said bacteriophage, pseudovirion, or phagemid, and a host cell, including recognition of and possibly binding to the natural receptor, whereby this interaction is followed by introduction of the genetic material of the bacteriophage into the host cell.
Artificial ligand: protein, protein domain or protein complex of the bacteriophage, pseudovirion, or phagemid, whereby said protein domain, protein, fusion protein or protein complex does not contain one or more peptide fragments of at least 10 contiguous amino acids derived from the natural ligand of the bacteriophage in the protein sequence or region that is involved in the interaction between bacteriophage, pseudovirion or phagemid and the artificial receptor.
Host cell: any bacterial cell that can allow a bacteriophage, pseudovirion or phagemid to enter said cell after interaction of a said bacteriophage, pseudovirion or phagemid with a natural or artificial receptor. As example, host cells include gram-negative or gram-positive bacteria, especially including E coli cells and in particular Fxe2x88x92cells which do not permit entering of bacteriophages, pseudovirions or phagemids through the pillin mechanism.
Entering bacteria: means that the bacteriophage, pseudovirion or phagemid can enter as a whole or as a part (e.g. only the genetic material) the host cell after specific binding to the artificial receptor. The mechanism by which the material is entering the host cell is not limited to specific ways and can be amongst others an active infection process or a passive uptake by the host cell. Methods for determination of the specific binding of the artificial ligand with the artificial receptor are illustrated in the examples.
Specific binding: means that the initial step of the entering is mediated by a specific interaction between the artificial receptor on the host cell wall and the artificial ligand of the bacteriophage, pseudovirion or phagemid. This specific interaction is preferentially a proteinxe2x80x94protein interaction. This entering after specific interaction should be distinguished from the Calcium dependent pilus independent infection that can be detected with M13 bacteriophages in which the second N-terminal domain of gIIIp has been removed (Krebber et al., 1997, J. Mol. Biol. 268: 607-618).
According to particular embodiments, the invention relates to a genetically modified host cell, transformed with a nucleotide sequence encoding an artificial receptor in conditions enabling that the artificial receptor be expressed at the surface of the host cell, said host cell being further transformed with a nucleotide sequence encoding said artificial ligand whereby said nucleotide sequence encoding the ligand entered the host cell as a consequence of the interaction between said artificial ligand and a protein sequence or region on said artificial receptor.
Particular genetically modified host cells are those wherein the nucleotide sequence encoding the artificial receptor and/or the nucleotide sequence encoding the artificial ligand are not initially known.
According to another specific embodiment, the genetically modified host cell is a gram-negative bacterium, especially an E coli cell of the Fxe2x88x92type.
According to another particular embodiment, the genetically modified host cell is a transformed cell wherein the nucleotide sequences of the artificial receptor and the nucleotide sequence for the artificial ligand are respectively coding sequences of an antibody or a functional fragment thereof and coding sequence of an antigen, or are respectively coding sequences of an antigen and coding sequence of antibody or a functional fragment thereof.
In said genetically modified host cell of the invention, the functional antibody fragment can be a variable fragment of an antibody, encompassing four-chain antibodies or two-chain antibody as defined in international patent application WO 94/04678, including native or modified, especially truncated chains thereof. In a preferred embodiment the variable chain is a VHH fragment of a camelid antibody or a functional portion of said VHH, as disclosed in the above cited patent application which is incorporated by reference.
The invention relates also to the above defined genetically modified host cell, wherein the nucleotide sequence encoding the artificial receptor comprises a sequence encoding Oprl or a part of Oprl sufficient to enable the exposure, at the surface of the host cell, of a protein sequence or region capable of interacting with the artificial ligand.
A further object of the invention is a kit comprising a genetically modified host cell according to the above proposed definitions and specific embodiments or comprising a host cell and/or a bacteriophage, pseudovirion or phagemid and/or means including a cloning vector enabling the construction of said host cell and/or a bacteriophage, pseudovirion or phagemid according to the above definitions. A particular kit is designed to be used for in vivo panning of antibody or antibody fragment library, or antigenic sequences library.
Said kit can also be used for the simultaneous in vivo panning of both an antibody fragment library, and an antigenic""sequences library.
The invention therefore provides means for the identification of target sequences or molecules including especially amino acid sequences capable of interacting with a determined receptor, whether the nature or sequences of said receptor is known or unknown. Especially the invention can be used for the identification of therapeutic targets.